Winkler E, Wachter E, Klingenberg M
Institute for Physical Biochemistry, University of Munich, Germany.
Biochemistry. 1997 Jan 7;36(1):148-55. doi: 10.1021/bi962178x.
The transport inhibiting nucleotide binding to the uncoupling protein (UCP) has a unique pH dependence and has been postulated to be controlled by the dissociation state of a carboxyl group in UCP with pK 4.5 and, in addition only for the nucleoside triphosphate, by a group with pK 7.2. To prove this assumption and to identify the carboxyl group, Woodward reagent K (WRK) was applied to UCP. In mitochondria, WRK was found to inhibit binding of GTP in a noncompetitive manner using WRK in the millimolar range. In isolated UCP, GTP binding is inhibited by WRK at a 1 to 2 ratio to UCP, suggesting that WRK primarily reacts with only one carboxyl group. Prebound GTP protects against WRK reaction as monitored by GTP binding. The protection decreases from pH 5 to 7 due to better reactivity of WRK and less tight GTP binding. WRK does not inhibit H+ transport by UCP but prevents GTP inhibition of H+ transport. For elucidating the WRK target residue, the WRK derivatized group was labeled with [3H] by reduction with [3H]NaBH4. Both GTP and GDP largely protected against WRK-dependent [3H] labeling. CNBr fragmentation identified the region T121-M197 as the [3H] incorporation site. Combined CNBr and tryptophane cleavage by the reagent 3-bromo-3-methyl-2-((2-nitrophenyl) thio)-3H-indole (BNPS) allowed to further delimit the 2.8 kDa peptide W173-M197 as the [3H] label carrier which contains two acid residues E190 and D195. To further identify the residue, limited tryptic digestion in sarcosyl-treated UCP was performed, and a tryptic fragment enclosing E190 and D195 was isolated which carried most of the [3H] label. Edman degradation showed the major [3H] label at the eighth position corresponding to E190 and no peak at D195. Thus, the original postulate of the pH-sensing carboxyl group regulating both the nucleoside di- and triphosphate binding has been verified. It is identified as E190 situated in the fourth transmembrane helix. In total, now four residues close to the nucleotide binding sites in UCP have been determined.
与解偶联蛋白（UCP）结合的转运抑制性核苷酸具有独特的pH依赖性，据推测它受UCP中一个pK为4.5的羧基解离状态控制，此外，仅对于核苷三磷酸，还受一个pK为7.2的基团控制。为了验证这一假设并确定羧基，将伍德沃德试剂K（WRK）应用于UCP。在线粒体中，发现毫摩尔浓度的WRK以非竞争性方式抑制GTP的结合。在分离的UCP中，WRK以1:2的比例抑制GTP与UCP的结合，这表明WRK主要仅与一个羧基反应。如通过GTP结合监测到的，预先结合的GTP可保护UCP免受WRK反应的影响。由于WRK的反应性增强和GTP结合变弱，这种保护作用从pH 5降低到pH 7。WRK不抑制UCP介导的H⁺转运，但可防止GTP对H⁺转运的抑制。为了阐明WRK的靶标残基，用[³H]NaBH₄还原使WRK衍生化的基团用[³H]标记。GTP和GDP都能很大程度地保护UCP免受WRK依赖性的[³H]标记。溴化氰裂解确定T121 - M197区域为[³H]掺入位点。结合溴化氰裂解和试剂3 - 溴 - 3 - 甲基 - 2 - （（2 - 硝基苯基）硫代）- 3H - 吲哚（BNPS）对色氨酸的裂解，可进一步将2.8 kDa的肽段W173 - M197确定为[³H]标记载体，该肽段包含两个酸性残基E190和D195。为了进一步确定该残基，在肌氨酸处理的UCP中进行了有限的胰蛋白酶消化，并分离出一个包含E190和D195的胰蛋白酶片段，该片段携带了大部分[³H]标记。埃德曼降解显示主要的[³H]标记在对应于E190的第八位，而在D195处没有峰。因此，最初关于调节核苷二磷酸和三磷酸结合的pH敏感羧基的假设得到了验证。它被确定为位于第四个跨膜螺旋中的E190。总共，现已确定UCP中靠近核苷酸结合位点的四个残基。
